Autophilic antibodies and method of making the same

ABSTRACT

Antibodies having noncovalent, autophilic properties are disclosed. The autophilic antibodies are derived from antibodies conjugated with an autophilic peptide. Such autophilic antibodies can promote apoptosis of target cells and enhance therapeutic efficacies in the treatment of patients with diseases or disorders responsive to antibody therapy. Compositions containing the antibodies, and methods of making and using the antibodies are also disclosed.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/481,248, filed Jun. 9, 2009, which is a continuation of U.S. patentapplication Ser. No. 11/119,404, filed Apr. 29, 2005, now U.S. Pat. No.7,569,674, which is a continuation-in-part of U.S. patent applicationSer. No. 10/652,864, filed Aug. 29, 2003, now abandoned, which claimspriority from U.S. Provisional Patent Application Ser. No. 60/407,421,filed Aug. 30, 2002. U.S. patent application Ser. No. 11/119,404 is alsoa continuation-in-part of U.S. patent application Ser. No. 09/865,281,filed May 29, 2001, now abandoned, which is a continuation-in-part ofU.S. patent application Ser. No. 09/070,907, filed May 4, 1998, now U.S.Pat. No. 6,238,667, which claims priority from U.S. Provisional PatentApplication Ser. No. 60/059,515, filed Sep. 19, 1997. The entire contentof each of these applications and patents is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to antibodies, compositions containingantibodies, and methods of using the antibodies and compositions in thetreatment of a variety of diseases, including those diseases treatablewith passive antibody therapy.

BACKGROUND OF THE INVENTION

Antibodies have emerged as a major therapeutic tool for the treatment ofchronic diseases such as cancer and autoimmune disorders. One of theprincipal advantages of these biological agents lies in their ability totarget disease-causing cells or molecules, while sparing healthy tissueand normal products of the body. However, antibodies that exhibitdesired specificities often fail in pre-clinical and clinicalevaluations because of inefficient targeting and/or low therapeuticactivity.

A rare class of antibodies, known as SuperAntibodies, exist in nature.These are antibodies that exhibit one or more properties not usuallyassociated with antibodies (Kohler H., et al., 1998; Kohler H., 2000).The defined class of SuperAntibodies comprises catalytic,membrane-penetrating, and autophilic antibodies and includes manyantibodies exhibiting superior targeting and therapeutic properties. Oneexample of a naturally occurring SuperAntibody is the murine TEPC-15antibody. TEPC-15 is an autophilic antibody which targets a normallycryptic determinant of phosphorylcholine on apoptotic cells andatheroschlerotic lesions. TEPC-15 antibodies have high therapeuticefficacy due to their ability to form dimers or multimers (on cell orbacteria surfaces, after binding to antigen), which enhances apoptosis.TEPC-15 antibodies are able to form dimers and multimers due to anautophilic peptide sequence. (Kang, C—Y, et al., 1988)

It is known that a major mechanism by which therapeutic antibodiesattack their target cells is through the induction of apoptosis.Apoptosis is triggered by crosslinking cellular receptors that are partof the apoptosis signal pathway. For example, crosslinking the B-cellantigen receptor by means of antibodies induces apoptosis in B-celltumors (Ghetie M., et al., 1997). Crosslinking of cellular receptorsalso increases the binding avidity of an antibody to its target antigen,and thus is likely to increase all cell surface-dependent therapeuticmechanisms, such as complement-mediated killing and complement-dependentopsonization and phagocytosis, antibody-dependent cellular cytotoxicity(ADCC), as well as enhanced inhibition of cell growth or alterations inmetabolic pathways within cells through increased binding to andblockade of cellular receptors when using antibodies targeted tocellular receptors.

To enhance the therapeutic efficacy of known antibodies, others haveproposed the use of hybrid molecules for therapeutic purposes whereinthe hybrid molecules comprise two distinct domains covalently linked.For instance, U.S. Pat. No. 6,482,586 (issued to Arab et al.) proposescovalent hybrid compositions for use in intracellular targeting. U.S.Pat. No. 6,406,693 (issued to Thorpe et al.) proposes antibodies andconjugates for killing tumor vascular endothelial cells by binding toaminophospholipid on the luminal surface.

These are but a few of the approaches that have been used to enhancetherapeutic efficacy of monoclonal antibodies that, in their native or“humanized” state, are not effective in killing their targets ortriggering a biological function affording therapeutic efficacy.

There is a need for a method of enhancing the therapeutic efficacy ofantibodies which have desired specificities without the use of toxicagents.

SUMMARY OF INVENTION

The present invention is directed to autophilic antibodies, compositionscontaining autophilic antibodies, methods of making autophilicantibodies, methods of restoring autophilic activity to antibodies thathave lost that activity, methods of assaying target antigens forautophilic antibodies, methods of enhancing apoptosis, complementfixation or cell-mediated killing using the autophilic antibodies, andmethods of using the autophilic antibodies and compositions in thetreatment of various diseases responsive to antibody therapy. Thediseases include those treatable with passive antibodies, includingatheroschlerosis, cancers, autoimmune disorders, Alzheimer's disease andother neuro-degenerative conditions, as well as artifacts of afunctioning immune system such as graft or transplant rejection.

The present invention relates to antibodies having autophilic propertiesthat mimic those of rare, naturally occurring, autophilic antibodies.Autophilic antibodies according to the present invention have theunusual property of spontaneously binding to one another after firstbinding to their target antigen (differential oligomerization).

The antibodies can comprise any antibody conjugated with an autophilicpeptide sequence. In some embodiments, the antibodies are capable ofbinding an antigen, which, when bound, has a therapeutic effect on adisease state or disorder. In some specific embodiments, the antibodiescomprise 5D10, S1C5, anti-caspase antibodies, anti-CD20 antibodies suchas rituximab, IF5, and tositumomab, anti-GM2 antibodies, humanized S107,trastuzumab, humanized TEPC-15, and humanized R24.

The antibodies can be conjugated with any autophilic peptide whichallows the antibodies to dimerize or oligomerize once bound to anantigen. The peptide can comprise any autophilic peptide sequence. Inspecific embodiments, the peptide comprises the T15 peptide sequence,the T15-scr2 peptide sequence, the R24 peptide sequence, the R24-chargedpeptide sequence, and optimized versions thereof.

Autophilic antibodies can also be conjugated with one or more otherpeptides to add additional functionality. In one embodiment, theautophilic antibodies can be conjugated to an autophilic peptidesequence and a transmembrane peptide sequence which allows theautophilic antibodies to penetrate inside cells and bind tointracellular targets. In specific embodiments, the transmembranepeptide sequence comprises MTS peptide or MTS-optimized peptide.

The invention also relates to compositions containing one or moreautophilic antibodies of the invention and pharmaceutically acceptablecarriers. The compositions can be administered to patients in need oftreatment with the autophilic antibodies of the invention. Thecompositions can be optimized to prevent the autophilic antibodies fromforming spontaneous dimers before administration.

The antibodies and compositions containing antibodies of the inventioncan be administered in doses similar to, or lower than, thosepracticable for non-autophilic antibodies.

The autophilic antibodies of the invention are preferably formed by oneof several methods, including chemically crosslinking a peptide capableof self-binding to an antibody. In a specific embodiment, the peptide iscross-linked to an antibody through oxidation of an N-linkedcarbohydrate. Alternatively, the autophilic peptide can be linked to anantibody through the nucleotide binding site or to a tryptophane bindingsite, or through less specific methods, such as through antibody epsilonamino groups or sulfhydryl groups obtained through partial reduction ofthe antibody.

The invention also relates to a method of optimizing autophilic peptidesequences for use in forming autophilic antibodies comprising optimizinga template-peptide.

The invention also relates to a method of restoring autophilicproperties to an antibody, such as a humanized antibody, which has lostits autophilic properties, in whole or in part, during the humanizationprocess, by conjugating an autophilic peptide to the antibody asdescribed above.

The invention also contemplates a method for assaying target antigensfor autophilic antibodies, and a method of testing the efficacy ofautophilic antibodies using animal models.

The invention also relates to methods of enhancing apoptosis, complementfixation, or cell-mediated killing using the autophilic antibodies ofthe invention comprising administering the antibodies of the invention.

The invention also relates to a method of treating a patient sufferingfrom a disorder, disease, or condition responsive to passive antibodytherapy comprising administering an autophilic antibody of the inventionto the patient.

BRIEF DESCRIPTION OF DRAWINGS

In drawings which are intended to illustrate embodiments of theinvention:

FIG. 1 is a graph depicting improved binding of anti-CD20 antibodiesconjugated with T15 peptide to DHL-4 cells at high concentrations ofantibody;

FIG. 2 is a graph depicting improved binding of anti-CD20 antibodiesconjugated with T15 peptide at low concentrations of antibody;

FIG. 3 is a graph depicting enhanced binding of anti-CD20 antibodiesconjugated with T15 peptide;

FIG. 4 is a graph depicting enhanced induction of apoptosis of tumorcells with mouse anti-CD20 conjugated with T15 peptide;

FIG. 5 is a graph depicting enhanced apoptosis of tumor cells usinganti-GM2 antibody conjugated with T15 peptide;

FIG. 6 is a graph comparing the efficacy of autophillic peptideconjugation to an affinity site on an antibody (nucleotide) versus anon-affinity site (CHO—carbohydrate) using anti-GM2;

FIG. 7 is a graph comparing the internalization of MTS conjugatedantibodies and non-MTS conjugated antibodies using anti-caspase 3antibodies;

FIG. 8 is a graph comparing the binding of Herceptin (upper panel) andthe autophilic peptide conjugated form of Herceptin (lower panel) tosmall cell lung cancer cells

FIG. 9 is a graph comparing the binding of anti-GM2 antibody and T15conjugated anti-GM2 antibody to ganglioside GM2;

FIG. 10 is a graph illustrating the self-binding activity of anti-GM2antibody and T15 conjugated anti-GM2 antibody;

FIG. 11 is a graph demonstrating binding specificity of T15 conjugatedanti-GM2 antibody to different gangliosides;

FIG. 12 is a graph depicting differences in cell surface binding ofanti-GM2 antibody and T15 conjugated anti-GM2 antibody to Jurkat cells;

FIG. 13 is a graph depicting the effect of anti-GM2 antibody and T15conjugated anti-GM2 antibody on Jurkat cell growth; and

FIG. 14 is a graph depicting the effect of chemotherapeutic drug(actinomycin D) on cell death in the presence and absence ofMTS-conjugated (Sab) antibody.

DETAILED DESCRIPTION OF THE INVENTION

Throughout the following description, specific details are set forth inorder to provide a more thorough understanding of the invention.However, the invention may be practiced without these particulars. Inother instances, well known elements have not been shown or described indetail to avoid unnecessarily obscuring the invention. Accordingly, thespecification and drawings are to be regarded in an illustrative, ratherthan a restrictive, sense.

The present invention relates to non-covalent, autophilic antibodieshaving enhanced therapeutic potencies. Such antibodies are referred toas “autophilic” antibodies. Autophilic antibodies belong to the class ofSuperAntibodies—antibodies that exhibit one or more properties notusually associated with antibodies (Kohler H., et al., 1998; Kohler H.,2000). The defined class of SuperAntibodies comprises catalytic,membrane-penetrating, and autophilic antibodies and includes manyantibodies exhibiting superior targeting and therapeutic properties.

The autophilic antibodies of the present invention comprise antibodiesconjugated with a peptide having an autophilic sequence. The autophilicantibodies of the invention can comprise any antibody. In someembodiments, the antibodies bind to targets implicated in a disease ordisorder, where binding of the target has a therapeutic effect on thedisease or disorder. The target antigens can include cell-surfaceantigens, including trans-membrane receptors. In specific embodiments,the antibodies comprise the monoclonal antibody 5D10, which binds humanB-cell receptors, the monoclonal antibody S1C5, which binds murineB-cell receptors, anti-CD20 antibodies such as rituximab, which bindsCD₂₀ on normal and malignant pre-B and mature B lymphocytes, mousemonoclonal antibody IF5, which is specific for CD-20 on human B-celllymphomas 5D10 and 3H1, and tositumab which also binds CD20 on Blymphocytes, anti-GM2, which binds human ganglioside GM2 lymphocytes,trastuzumab, which binds the protein HER2 that is produced by breastcells, anti-caspase antibodies, which recognize the caspase proteinsinvolved in apoptosis, humanized TEPC-15 antibodies, which are capableof binding oxidized low density lipoproteins (oxLDL) and can preventuptake of oxidized LDL by macrophages, humanized T15-idiotype positiveantibodies, which bind phosphocholine, and humanized R24 antibodieswhich recognize the human GD3 ganglioside on melanoma cell surfaces.

The autophilic antibodies of the invention are conjugated with anautophilic peptide component. The autophilic peptide can comprise anyautophilic peptide sequence. The autophilic peptide can also compriseoptimized sequences which may include sequences with enhancedfunctionality, such as ones which act as linkers to enhance display andcross-linking activity of antibodies, or residues which enhancesolubility of autophilic sequences. In all situations, the autophilicsequences are complementary and are able to bind to themselves.

In a specific embodiment, the autophilic peptide comprises theautophilic T15 peptide, which originally comprised regions of CDR2 andFR3 of the murine germline-encoded S107/TEPC15 antibody. The T15 peptidecomprises amino acid sequence: ASRNKANDYTTDYSASVKGRFIVSR (SEQ ID NO.: 1)(Kang C-Y, et al., 1988). Its autophilic property has been shown to beantigen-independent. Therefore, attachment of the peptide to anymonomeric antibodies can impart autophilic and increased avidityproperties to the antibodies (Y. Zhao, and H. Kohler, 2002). In otherspecific embodiments, the autophilic peptide can comprise a humanizedT15 peptide sequence, for increased or optimized binding andeffectiveness of antibodies.

In other specific embodiments, the autophilic peptide can also comprisethe peptide T15-scr2 comprising the sequenceNH-SKAVSRFNAKGIRYSETNVDTYAS-COOH (SEQ ID NO. 4), the peptide R24comprising the sequence NH-GAAVAYISSGGSSINYA-COOH (SEQ ID NO. 5), thepeptide R24-Charged comprising the sequence NH-GKAVAYISSGGSSINYAE-COOH(SEQ ID NO. 6), and any modifications to the peptides which optimize orenhance the binding and therapeutic effectiveness of antibodies.

The autophilic antibody conjugates of the invention can also compriseone or more other bioactive or functional peptides which conferadditional functionality on the antibody conjugates. For example, theantibody conjugate can comprise an antibody that bears a T15 autophilicpeptide and an MTS membrane translocation peptide (Y. Zhao et al., 2003;Y. Lin et al., 1995). In a specific embodiment, the MTS translocationpeptide can have the amino acid sequence KGEGAAVLLPVLLAAPG (SEQ ID NO.2). In another embodiment, the translocation peptide can be an optimizedMTS peptide, MTS-optimized, comprising the sequence WKGESAAVILPVLIASPG(SEQ ID NO. 7). The T15 peptide provides autophilicity to the conjugate,and the MTS sequence allows the antibody to penetrate into cells. Such aconjugate can target, for example, cancer cells for radio-immunotherapy,when its antibody region targets a primarily intracellular,tumor-associated antigen, such as carcino-embryonic antigen (CEA) (See,e.g., U.S. Pat. No. 6,238,667 which is hereby incorporated byreference). The autophilic conjugate, upon administration, targetsCEA-bearing, colon carcinoma cells, is internalized by translocation ofthe antibody mediated by the MTS peptide, and is enabled to bind to themore prevalent intracellular form of CEA. Crosslinking of CEA antibodywith, for instance, a therapeutic isotope such as ¹³¹I will be retainedin a cell longer than unmodified, labeled antibody and will deliver ahigher radioactive dose to the tumor. In addition, such therapeuticisotopes as ¹²⁵I, which release beta particles of short path length andare not normally considered useful for therapy, can, when deliveredintracellularly in closer proximity to the nucleus, be efficaciousagainst certain targets, especially those of lymphoid origin andaccessible in the blood and lymph tissues. Other categories ofsecondary, bioactive or functional peptides include peptides capable ofbinding to receptors, and peptide mimetics, capable of binding to adistinctive antigen or epitope of the same antigen, targeted by theprimary antigen combining site.

Autophilic antibodies conjugated with one or more other functionalpeptides may also be useful for targeting intracellular antigens. Suchantigens could include tumour associated antigens and viral proteins.For example, an autophilic antibody specific for viral proteins which isconjugated with a self-binding peptide and a MTS peptide can also beused to bind to intracellular viral proteins and prevent production ofviruses. The antibody could be internalized through the MTS peptide, andwould be optimized to bind intracellular viral proteins (Zhao, Y., etal. 2003). Many other functional peptides may also be conjugated to theautophilic antibodies to increase functionality.

The invention also relates to compositions containing the autophilicantibodies of the invention and a pharmaceutically acceptable carrier.The conjugate autophilic antibodies can bind non-covalently with otherautophilic antibodies when bound to their target antigen(s). However,premature formation of dimers or multimers of the antibodies may lead todifficulties in manufacturing, such as during purification andconcentration, as well as drawbacks in administration, which may lead toside effects. As such, compositions containing the autophilicantibody-peptide conjugates of the invention are formulated to reducethis dimerizing potential and maximize monomericity while in solutionand before administration. For example, it has been found that solutiondimerization can be reduced or mitigated by using a hypertoniccomposition. In some embodiments, salt concentrations of 0.5M or more,low levels of SDS or other various detergents such as those of ananionic nature (see U.S. Pat. No. 5,151,266 which is hereby incorporatedby reference), or modifications of the antibody to decrease itsisoelectric point, for example through the use of succinyl anhydride(see U.S. Pat. No. 5,322,678, which is hereby incorporated byreference), can be used to formulate compositions.

According to the principles of the present invention, an autophilicantibody or a composition containing an autophilic antibody ispreferably administered in one or more dosage amounts substantiallyidentical to, or lower than, those practicable for unmodifiedantibodies. Thus, in the treatment of a lymphoma or a breast cancer, anautophilic antibody of the invention can be administered in one or moredose amounts substantially identical to, or less than, the doses usedfor RITUXAN™ (rituximab) or HERCEPTIN™ (trastuzumab). For example,treatment with HERCEPTIN™ (a humanized monoclonal anti-HER2/neuantibody) in a patient with HER2⁺ breast cancer employs an antibodyconcentration of about 10 mg/ml. Intravenous infusion over 90 minutesprovides a total dose of 250 mg on day 0. Beginning at day 7, 100 mg isadministered weekly for a total of 10 doses. The dosing regimen isreduced gradually from 250 mg to 100 mg to a maintenance dose of 50 mg.Similar or lower dosage regimens to that for HERCEPTIN™ can be employedwith autophilic antibodies, with any adjustments being well within thecapabilities of a skilled practitioner.

The present invention also relates to a method of producing theautophilic antibody conjugates. The antibody conjugates can be producedby chemical or genetic engineering techniques. For instance, a peptidecomponent of an autophilic antibody can be attached to theimmunoglobulin component via its variable domain structures usingazido-tryptophan or azido-purine photoactivation crosslinking. In thisapproach, the peptide attaches to the variable domain at a location thatdoes not interfere with antigen recognition. This method can incorporatetwo peptide moieties into a single immunoglobulin molecule. See, forexample U.S. Pat. No. 6,238,667, U.S. Reissue Pat. No. RE38,008, U.S.Pat. No. 5,635,180, and U.S. Pat. No. 5,106,951, the disclosures ofwhich are incorporated herein by reference.

The peptides can be photo-crosslinked to a heterocyclic compoundaffinity site (such as a tryptophane affinity site) or a nucleotideaffinity site of antibodies to produce the autophilic antibodies of theinvention. Alternatively, the peptides can be crosslinked to acarbohydrate site of the Fc portion or to an amino or sulfhydryl groupof an antibody. In an alternative embodiment, the autophilic antibodycan be conveniently expressed as a fusion protein of the autophilicpeptide and whole immunoglobulin, or fragment thereof.

The present invention also contemplates a method of producing anautophilic conjugate of the invention in which a template peptide hasbeen modified to enhance the crosslinking potential of the autophilicantibodies as described above. In one embodiment of the invention, suchfunctionally enhanced peptides are determined by producing a series ofsynthetic peptides with substitutions at each amino acid position withinthe template sequence and then testing this library of peptides forautophilic binding or for binding to the original peptide sequence.Those peptides with superior binding to the original sequence are thenconjugated to immunoglobulins and the resultant conjugates are testedfor potency, specifity, and the unwanted ability to induce aggregation.In one specific embodiment, the T15 peptide sequence is altered andmodified sequences are selected for enhanced function.

In other embodiments of the invention, the self-binding potential of apeptide can be enhanced by increasing complementarity of the sequence,such as described in U.S. Pat. No. 4,863,857 to Blalock et al., which isincorporated herein by reference. The self-binding potential of apeptide can also be enhanced by humanizing a self-binding peptidesequence which is derived from non-human animals. Humanizing a peptidesequence involves optimizing the sequence for expression orfunctionality in humans. Examples and methods of humanizing peptides andproteins have been previously described (Roque-Navarro et al., 2003;Caldas et al., 2003; Leger et al., 1997; Isaacs and Waldmann, 1994;Miles et al. 1989; Veeraraghavan et al., 2004; Dean et al., 2004;Hakenberg et al., 2003; Gonzales et al., 2004; and H. Schellekens,2002).

An assay method is also contemplated that permits pre-selection oftarget antigens most suitable as targets for the autophilic antibodiesof the present invention. Such method entails the in vitro assay ofapoptosis with multiple antigen-positive target cell lines, and ifpossible, fresh isolates of antigen-positive cells. A non-modifiedantibody is incubated with a secondary (anti-immunoglobulin) antibody toenhance the potential for cross-linking. Cells may be enumerated bypre-labeling, such as with ⁵¹Cr or ¹³¹I-UDR, or by FACS analysis usingindicators of apoptosis. Positive results in this assay predict apositive outcome using an autophilic conjugate. However, negativeresults in the assay do not necessarily mean that subsequent conjugationwith an autophilic peptide will not improve one or more antibodyeffector properties.

Autophilic antibodies of the present invention have a higher potentialfor forming dimers in vitro under laboratory conditions, such as insolution with PEG. This laboratory characteristic correlates with acrosslinking ability upon binding to a cell-surface target and highertherapeutic potency through such mechanisms as triggering apoptosis.This characteristic can be used to identify natural SuperAntibodies andto screen for proper conjugation of self-binding peptides to anon-autophillic antibody.

A method of enhancing apoptosis, complement fixation, effectorcell-mediated killing of targets, or preventing the development of, orenhancement of, a disease state, is also disclosed employing anautophilic conjugate of the invention or a composition comprising anautophilic conjugate of the invention. In one embodiment, an autophilicconjugate of the invention, or a composition containing an autophilicconjugate of the invention, is administered to a subject. Onceadministered, the antibodies bind to target cells and enhance apoptosis,complement fixation, effector cell-mediated killing of targets, orprevent target antigens or cells from stimulating the development of, orfurther enhancing, a disease state. In a further embodiment, allowingtime for the autophilic conjugate to bind to target cells and enhanceapoptosis, complement fixation, effector cell-mediated killing oftargets, or prevent target antigens or cells from further enhancing adisease state, and for the autophilic conjugate to be cleared fromnormal tissues, a second anti-autophilic peptide antibody can beadministered. For example, if an autophilic conjugate contains anon-native autophilic peptide, such as the murine T15 sequence, ananti-T15 peptide antibody would be administered, which would onlyrecognize and bind to antibodies conjugated with the T15 sequence. Thisallows binding to and enhancement of apoptosis of pre-localizedSuperAntibodies.

A further method of enhancing apoptosis, complement fixation, oreffector cell-mediated killing of targets is contemplated, which employsadministering an autophilic conjugate of the invention in which atemplate autophilic peptide has been modified to enhance thecrosslinking potential of the autophilic antibodies as described above.

In another aspect of the invention, a method of potentiating apoptosisof targeted cells of a patient comprises administering a firstautophilic antibody-peptide conjugate, or a composition containing anautophilic antibody-peptide conjugate, and a second antibody, orcomposition containing the second antibody, that recognizes theautophilic peptide domain of the conjugate. In this embodiment, theantibody-peptide conjugate recognizes an antigen on a target cell. Owingto its homodimerization property, the antibody-peptide conjugate canbind more avidly to the target than the corresponding antibody lackingthe autophilic peptide domain. This is likely due to the ability tocrosslink antigen at the surface of target cells. Moreover, whenever theautophilic antibodies bind to two or more antigens, with those antigensbeing brought in close proximity and crosslinked, due to the autophilicproperty of the antibodies, an apoptosis signal within the cell can betriggered. In those instances when the peptide domain of the conjugatepresents an exposed epitope, a second antibody, specific for theautophilic peptide, can be administered, bind to the modified antibody,and enhance the process of crosslinking and even cause temporaryclearance of the target antigen. As an example, if the target antigen isa receptor, clearance from the cell surface, endocytosis, anddegradation will subsequently require synthesis of new receptor protein,meaning that the biological function of the receptor will be moreeffectively inhibited for a longer period than using either a simpleblocking antibody or small molecule inhibitor. Alternatively, the secondantibody can bear a radiolabel or other potentially therapeuticsubstance, so that when administered, it can attack the targeted cells.The key to use of this second antibody is the antibody's specificity.The autophilic peptide is present on only a small number ofimmunoglobulins, or if it is a peptide derived from another organism, orif it is modified, will not be present on any immunoglobulins in apatient. Thus, antibody specific to the autophilic peptide will have therequisite selectivity to be used in vivo.

In another aspect of the invention, a patient who suffers from a diseaseor condition responsive to antibody therapy is administered at least oneautophilic antibody of the invention in an amount effective to alleviatesymptoms of the disease or condition. A disease or conditioncontemplated for treatment by an antibody of the invention can be amalignancy, neoplasm, cancer, auto-immune disorder, Alzheimer's diseaseor other neuro-degenerative condition, graft or transplantationrejection, atherosclerosis, or any other disease or condition responsiveto antibody therapy.

The following examples are presented to illustrate certain aspects ofthe invention, and are not intended to limit the scope of the invention.

EXAMPLES Example 1 Conjugation of T15 Peptide to two Mabs Specific forB-Cell Receptor

Cell Line and Antibodies

The human B-cell tumor line (Su-DHL4) and murine B-cell tumor line(38C13) are grown in RPM 1640 medium (supplemented with 10% fetal bovineserum, 2 μmol/L glutamine, 10 μmol/L HEPES, 50 U/mL penicillin, and 50μg/mL streptomycin, 50 μmol/L 2-mercaptoethanol) at 37° C. under 5%carbon dioxide. Two mAb 5D10 and S1C5, specific for the human or murineBCR, respectively, were used in this study. The antibodies are purifiedfrom the culture supernatant by protein G and protein A affinitychromatography.

Synthesis of Antibody-Peptide Conjugate.

T15H peptide (ASRNKANDYTTDYSASVKGRFIVSR) (SEQ ID NO. 1), a VH-derivedpeptide from an autophilic antibody-T15, was synthesized by GenemedSynthesis (San Francisco, Calif., U.S.A.). Antibodies were dialyzedagainst PBS (pH 6.0) and 1/10 volume of 200 μmol/L sodium periodate wasadded and incubated at 4° C. for 30 minutes in the dark. The reactionwas stopped by adding glycerol to 30 μmol/L, and the sample was dialyzedat 4° C. for 30 minutes against PBS (pH 7.0). One hundred timesmolecular excess of T15H or scrambled peptide was added to theantibodies and incubated at 37° C. for 1 hour. L-Lysine was added andincubated at 37° C. for 30 minutes to block the remained aldehyde group.The same oxidation reaction steps (except adding the peptides) wereapplied to antibodies used as controls. After the blocking step, theantibody conjugates were dialyzed against PBS (pH 7.2) overnight.

Ig Capture ELISA.

Four μg/mL of SIC5-T15H was coated to Costar vinyl assay plates (Costar,Cambridge, Mass.). After blocking with 3% BSA solution, 8 μg/mL ofphotobiotinylated S1C5-T15H, S1C5-scrambled peptide conjugate, andcontrol S1C5 were added to the first wells, and 1:1 dilution wasperformed. The antibodies were incubated for 2 hours at roomtemperature. After washing with PBS buffer, Avidin-HRP (Sigma, St.Louis, Mo.) was added as a 1:2500 dilution. The binding antibodies werevisualized by adding substrate o-phenylenediamine.

Size Exclusion Chromatography.

Antibody conjugate was chromatographed on a 75 mL Sephacryl 300HR column(Pharmacia, Peapack, N.J.). 1:10 diluted PBS (pH 7.2) was chosen aselution buffer. Fractions (0.5 mL/each) were collected and aliquots (100μL) were assayed on antihuman IgG capture ELISA. The ELISA reading (OD490 nm) is plotted against elution volume.

Viability Assay for Antibody-Treated Cells.

The lymphoma cells were grown in 96-well tissue culture wells in 1-mLmedium. 2 μg of antibodies or antibody-peptide conjugates were added andincubated for various times as described herein. Ten μL aliquots fromthe cell suspension were used to determine viability by using trypanblue exclusion.

FACS Assay of the B-Cell Lymphoma.

The Su-DHL4 and 38C13 cells were fixed with 1% paraformaldehyde. 1×10⁶cells were suspended in 50 μL of staining buffer (Hank's balanced saltsolution, containing 0.1% NaN3, 1.0% BSA), then 1.5 μg ofphotobiotinylated S1C5-T15H conjugates was added and incubated for 30minutes on ice. Control antibodies and antibody-scrambled T15 peptideconjugates served as controls. The cells were washed twice with stainingbuffer before Avidin-FITC (Sigma) was added to the cells for 30 minuteson ice. Then the cells were washed twice with staining buffer,re-suspended in 200 μL PBS and analyzed by flow cytometry.

Hoechst-Merocyanin 540 Staining to Detect Apoptosis.

1×10⁶ of lymphoma cells were placed into 24-well tissue culture wells.Four μg of antibodies or antibody-peptide conjugates were added andincubated for various times as described herein. 1×10⁶ cells wereremoved from the culture, re-suspended in 900 μL cold PBS (pH 7.2). Onehundred μL of Hoechst 33342 (50 μg/mL; Molecular Probe, Eugene, Oreg.,U.S.A.) was added, the cells were incubated at 37° C. for 30 minutes inthe dark. The cells were centrifuged and re-suspended in 100 μL PBS.Then, 4 μL of MC540 solution (Molecular Probe) was added, and a20-minute incubation was performed at room temperature in the dark. Thecells were pelleted, re-suspended in 1 mL cold PBS (pH 7.2), andanalyzed by flow cytometry.

Results

Characterization of Autophilic Antibodies

The T15H (24-mer) peptide was crosslinked to two murine mAb (SIC5 and 5D10), using carbohydrate periodate conjugation. The mAb S1C5 (IgG1) isspecific for the tumor idiotype of the mouse 38C13 B-cell line and the5D10 antibody for the human Su-DHL4 B-cell tumor. Both antibodiesrecognize unique idiotypes of the BCR IgM on the B-cell tumors.

Autophilic Behavior can Easily be Demonstrated by ELISA.

The autophilic effect was studied with the T15H peptide-crosslinked mAbS1C15.

The T15H-crosslinked S1C5 binds to insolubilized S1C5-T15H detected bybiotin-avidin ELISA. Control SIC5 does not bind significantly toS1C5-T15H or S1C5 crosslinked with a scrambled peptide. Similarself-binding of T15H peptide-crosslinked mAb 5D10 to insolubilizedT15H-5D10 was also observed. The specificity of the peptide mediatedautophilic effect was tested using the 24-mer peptide T15H itself as aninhibitor. Only the T15H peptide inhibited S1C5-T15H and 5D10-T15Hself-binding while the control-scrambled peptide did not inhibit it.These results are similar to the previously published inhibition datawith the naturally occurring autophilic T15/S107 antibody (not shown).

T15H-Antibody Conjugates Form an Equilibrium of Monomer and Dimer inSolution.

The non-covalent nature of the self-aggregation of T15H-linkedantibodies raises the question of its physical state in solution. Toaddress this issue, the molecular species of T15H-linked monoclonalantibodies were analyzed using gel electrophoresis and sizing gelfiltration. The electrophoretic mobility of control and T15H peptideconjugated to SIC5 and 5D 10 under reducing and non-reducing conditionsshow no differences, indicating the absence of chemical bonds betweenthe antibody chains. The molecular species of the peptide-conjugatedantibodies (5D0-T15H) was further analyzed by size exclusionchromatography. The elution profile indicated two immunoglobulin speciesof different sizes. The larger first peak eluted in the position of anantibody dimer. The second smaller peak eluted in the position ofnon-conjugated 5D10 antibody. The appearance of two peaks resembledmonomer and dimer antibodies and could indicate that either a fractionof antibodies was not modified to polymerize, or that the modificationwas complete and the antibody establishes an equilibrium of dimers andmonomers. To test the latter possibility, material from both peaks weresubjected to a second gel filtration on the same column. Reruns of bothpeaks yielded again two peaks at the same position as in the firstchromatography (Zhao and Kohler, 2002). These data show that the T15Hpeptide-linked antibodies exist in solution as two distinct molecularspecies in equilibrium as monomer and dimer.

Enhanced Binding of Autophilic Antibodies to Tumors.

The binding of the peptide-conjugated antibodies against theirrespective tumor targets was compared with that of the controlantibodies in indirect fluorescence activated cell sorting (FACS). Ascontrol, antibodies linked with a scrambled peptide were included. Thefluorescence intensity of the T15H-S1C5 on 38C13 cells is compared withthat by the control S1C5 and the scrambled peptide S1C5. The differencein mean fluorescence channels between S1C5-T15H and controls was greaterthan 10-fold. Similarly, the FACS analysis of autophilic 5D 10-T15H onSu-DHL4 cells shows enhancement of binding over binding of control 5D10and control peptide-crosslinked 5D 10. In both tumor systems, theconjugation of the T15H peptide to tumor-specific antibody enhanced theFACS signals over control antibodies used at the same concentration(Zhao, Lou, et al., 2002). The enhancement of fluorescence can beexplained with the increase of targeting antibodies caused byself-aggregation and lattice formation on the surface of the tumorcells.

Inhibition of Tumor Growth.

Antibodies binding to the BCR induce crosslinking of the BCR, which, inturn, inhibits cell proliferation and produces a death signal.Furthermore, chemically dimerized antibodies directed against a B-celltumor induce hyper-crosslinking of the BCR followed by inhibition ofcell division and apoptosis of the tumor. To see if similar enhancementof the antitumor effects of dimerizing antibody were induced by ournoncovalent, dimerizing T15H-linked antibodies, the two B cell tumorswere cultured in the absence or presence of control and T15H-linkedantibodies. Co-culture of both tumors, 38C13 and Su-DHL4, with theirrespective T15H-linked antibodies inhibited the cell growthsignificantly better compared with the control antibodies. To test thetumor target specificity of autophilic antibodies in growth inhibition,criss-cross experiments were performed with the 38C13 and Su-DHL-4 celllines. Inhibition of 38C13 cell growth with S1C5-T15H was statisticallygreater than mismatched 5D10-T15H. Similar results on the specificity ofautophilic antibodies were obtained with the Su-DHL4 cells (Zhao, Lou,et al., 2002).

Induction of Apoptosis.

As suggested by earlier studies, the antitumor effect of antibodiesdirected against the BCR of B-cell lymphomas in vitro and in vivo mightbe caused by the induction of apoptosis. Aliquots of tumor cells (38C13and Su-DHL-4) cultured in the presence of control or T15H-linkedantibodies were analyzed for apoptosis using a double stain FACSprotocol. 38C13 and Su-DHL4 cells underwent a moderate amount ofapoptosis without antibodies over a 6, respectively 18-hour culture.This apoptosis was enhanced when the respective antibody was added.However, when the T15H-linked antibodies were added, the accumulatednumber of apoptotic 38C13 cells was almost doubled, and apoptosis ofSu-DHL4 cells was more than doubled during the entire culture (Zhao,Lou, et al., 2002).

The biologic advantage of the autophilic property is exemplified withthe S107/T15 anti-phosphorylcholine antibody. This autophilic antibodyis several times more potent in protecting immune-deficient mice againstinfection with pneumococci pneumoniae than non-autophilic antibodieswith the same antigen specificity and affinity.

As shown here, the autophilic antibody function can be transferred toother antibodies by chemically crosslinking a peptide derived from theT15 VH germline sequence. The modified antibody mimics the autophilicproperty of the T15/S107 antibody, producing a autophillic antibody withincreased avidity and enhanced targeting. Enhancing the binding ofautophilic engineered antibodies to the BCR of B-cell tumor increasesthe strength of the death signals leading to profound inhibition of cellproliferation in culture. Even though the doubling of apoptosis isdemonstrated here, other mechanisms of growth inhibition can beinvolved.

Crosslinking the BCR of the mature murine B-cell lymphoma A20 canprotect against CD95 mediated apoptosis. This anti-apoptotic activity ofengagement of the BCR by crosslinking antibodies is highly restricted tothe time window of CD95 stimulation and is not dependent upon proteinsynthesis. The finding that BCR hypercrosslinking per se ispro-apoptotic is not at variance with reports on the anti-apoptoticactivity of the BCR engagement, because it can be a result of the use ofless mature B-cell lines in our study, to different strength ofdelivered signals by homodimerizing antibodies, or to Fas-independentapoptosis.

The use of two BCR idiotope-specific antibodies against different tumorsoffered the opportunity to test the biologic effect of targetingreceptors other than the idiotope specific BCR. In criss-crossexperiments with autophilic antibodies binding in FACS analysis andinhibition of growth in vitro show a significant enhancement only withthe autophilic matched antibody. In this context, it is interesting tospeculate whether enhanced tumor targeting would also augment cellulareffector functions.

In an earlier study using chemically homodimerized antibodies, the Fedomain was not involved in the augmentation of growth inhibition andtumor cells lacking Fc receptors were susceptible to the antigrowthactivity of homodimers. Thus, the anti-tumor effect induced bydimerizing antibodies would not be restricted to lymphoid tumors such asnon-Hodgkin's B-cell lymphoma, where anti-tumor effects require theparticipation of Fc-receptor-bearing effector cells.

The described approach of transferring the naturally occurringautophilic property to other antibodies thereby enhancing theiranti-tumor effect outlines a general method to improve the therapeuticefficacy of antibodies in passive immunotherapy.

Example 2 Internalization of Antibodies Conjugated with MTS Peptide

Cell Line and Antibodies

Human Jurkat T cells were grown in RPMI 1640 supplemented with 10% fetalbovine serum and antibiotic (penicillin, streptomycin and amphotericin).Rabbit polyclonal anti-active caspase-3 antibody (#9661 S) and anticleaved-fodrin, i.e. alpha H spectrins (#2121S) were purchased from CellSignaling, Inc (Beverly, Mass.). Monoclonal (rabbit) anti-activecaspase-3 antibody (#C92-605) was purchased from BD PharMingen (SanDiego, Calif.). Mouse monoclonal antibody 3H1 (anti-CEA) was purifiedfrom cell-culture supernatant by protein G affinity chromatography.Anti-mouse and anti-rabbit HRP-conjugated secondary antibodies werepurchased from Santa Cruz Biotechnologies, Inc. ApoAlert Caspase-3Fluorescent Assay kit was purchased from Clonetech Laboratories (PaloAlto, Calif.). The Cell Death Detection ELISA was purchased from RocheApplied Science (Indianapolis, Ind.).

Synthesis of MTS Peptide-Antibody Conjugate

MTS peptide (KGEGAAVLLPVLLAAPG) (SEQ ID NO. 2) was a signalpeptide-based membrane translocation sequence, and synthesized byGenemed Synthesis (San Francisco, Calif.). Antibodies were dialyzedagainst PBS (pH 6.0) buffer, oxidized by adding 1/10 volume of 200mmol/L NaIO4 and incubating at 4° C. for 30 min in the dark. Addingglycerol to a final concentration of 30 mM terminated the oxidationstep. Samples were subsequently dialyzed at 4° C. for 1 h against 1×PBS(pH6.0) buffer. The MTS peptide (50 times molar excess) was added tocouple the antibodies and the samples were incubated at 37° C. for 1 hand the resulting antibody-peptide conjugate was dialyzed against 1×PBS(pH 7.4).

Effect of MTS-Conjugated Antibody on Cell Growth

Jurkat cells (2.5×10⁵) were seeded into 96-well culture plate. Afterincubation with 0.5 μg MTS-antibody conjugates for 6, 12, 18 and 24hour, aliquots were removed and viability was determined by trypan blueexclusion.

Study of Antibody Internalization by ELISA

Jurkat cells, grown in 1-ml medium in a 6-well culture plate, wereincubated with 2 μg of unconjugated or MTS conjugated antibodies for 0,1, 3, 6, 12 and 18 h. The cells were centrifuged and the culturesupernatant was then transferred to a new tube. The cell pellet waswashed twice with PBS (pH 7.4) before being homogenized by Pellet PestleMotor (Kontes, Vineland, N.J.) for 30 sec. All of the cell homogenateand an equal volume of the culture (10 μl) supernatant were added tosheep anti-rabbit IgG coated ELISA plate (Falcon, Oxnard, Calif.) andincubated for 2 h at room temperature. After washing step, HRP-labeledgoat anti-rabbit light chain antibody was added, and visualized usingo-phenylenediamine.

DNA Fragmentation

Jurkat cells were pre-treated with antibodies or a caspase-3 inhibitor(DEVD-fmk) for 1 h, centrifuged, and incubated with fresh mediumcontaining actinomycin D alone (1 μg/ml) for 4 h. After treatment,Jurkat cells were collected, washed, and resuspended in 700 μl of HLbuffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.2% Triton X-100^(4,11) for15 min at room temperature. DNA was extracted withphenol:chloroform:isoamyl alcohol (25:24:1) and precipitated 24 h at−20° C. with 0.1 volume of 5 M NaCl and 1 volumes of isopropanol. TheDNA was washed, dried, and resuspended in TE pH 8.0. The DNA wasresolved by electrophoresis on a 1.5% agarose gel and visualized by UVfluorescence after staining with ethidium bromide. DNA fragmentation wasalso determined using the Cell Death Detection ELISA according to themanufacturer's instructions.

Preparation of Total Cell Lysate

Jurkat cells were treated as described in the DNA fragmentation section.After treatment, cells were collected and washed with PBS (pH 7.4)twice, then suspended in 300 μl of CHAPS buffer (50 mM PIPES, pH 6.5, 2mM EDTA, 0.1% CHAPS). The samples were sonicated for 10 sec andcentrifuged at 14,000 rpm for 15 min at 4° C. The supernatant wastransferred to a new tube and referred as total cell lysate.

Caspase-3-Like Cleavage Activity Assay

Jurkat cells were treated as described in the DNA fragmentation section.Equal amounts of protein of the total cell lysate was applied forcaspase-3 activity assay using ApoAlert Caspase-3 Fluorescent Assay Kitaccording to the manufacturer's instruction. Fluorescence was measuredwith a Spectra MAX GEMINI Reader (Molecular Devices, Sunnyvale, Calif.).

Western Blot Analysis

Jurkat total cell lysates (10 ug) were separated on a 10% SDS-PAGE gelto detect immunoreactive protein against cleaved spectrin. Ponceaustaining was used to monitor the uniformity of protein transfer onto thenitrocellulose membrane. The membrane was washed with distilled water toremove excess stain and blocked in Blotto (5% milk, 10 mm Tris-HCl [pH8.0], 150 mM NaCl and 0.05% Tween 20) for 2 h at room temperature.Before adding the secondary antibody, the membrane was washed twice withTBST (10 mM Tris-HCl with 150 mM NaCl and 0.05% Tween 20), and thenincubated with horseradish peroxidase-conjugated secondary antibodies.The blot was washed extensively and reactivity was visualized byenhanced chemiluminescence (AmershamBiotech, Piscataway, N.J.).

Statistical Analysis

Statistical analysis was performed using the student t-test (for apair-wise comparison) and one-way ANOVA followed by Newman-Keulsposttest. Data are reported as means.+−.SE.

Results

As shown in FIG. 7, an MTS conjugated anti-active caspase 3 antibody isinternalized more rapidly than unmodified antibody. When cells wereexposed to the chemotherapeutic drug, actinomycin D, apoptosis wastriggered and the cells died (see FIG. 14). However, if cells wereexposed at the same time to the MTS conjugated antibody (transMab), mostof the toxicity of the chemotherapeutic drug was inhibited.

Example 3 Enhancing Binding and Apoptosis Using Peptide-ConjugatedAnti-CD20 Antibodies

Materials and Methods

Cell Line and Antibodies

The human B-cell tumor lines SU-DHL-4 and Raj were grown in RPMI 1640medium, supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 10μmol/L Hepes, 50 U/mL penicillin, 50 .mu.g/mL streptomycin, and 50.mu.mol/L 2-mercaptoethanol at 37° C. under 5% CO₂. Mouse monoclonalantibodies IF5 IgG2a (ATTC #HB-9645) specific for human B-cell lymphomas5D10 and 3H1 (Zhao, Lou, et al., 2002.) were purified from cell culturesupernatant by protein G or protein A affinity chromatography.

Synthesis of Antibody-Peptide Conjugate

T15 peptide (ASRNKANDYTTDYSASVKGRFIVSR) (SEQ ID NO. 1), a VH-derivedpeptide from a self-binding antibody-T15, was synthesized by GenemedSynthesis, California. The antibodies were dialyzed againstphosphate-buffered saline (PBS; pH 6.0) buffer; sodium periodide (1:10volume of 200 μmol/L NaIO4) was added; and the mixture was incubated at4° C. for 30 minutes in the dark. The oxidation was stopped by addingglycerol (30 .mu.mol, final concentration), and dialysis was performedat 4° C. for 30 minutes against PBS (pH 7.0) buffer. One hundred timesmolecular excess of T15 peptide was coupled to the antibody 1F5 byincubation at 37° C. for 1 hour; 1-lysine was used to block theunreacted aldehyde by incubation at 37° C. for 30 minutes. After theblocking step, the antibody conjugates were dialyzed against PBS (pH7.2) overnight.

8-azido-adenosine-biotin was synthesized and used to affinity cross-linkbiotin to antibodies. The 8-azidoadenosine dialdehyde was prepared aspreviously published (U.S. Pat. No. 5,800,991 for “Nucleotide ornucleoside photoaffinity compound modified antibodies, methods for theirmanufacture and use thereof as diagnostics and therapeutics,” issued toHaley et al., 1998, which is incorporated herein by reference)

Self-Binding Enzyme-Linked Immunosorbent Assay

Four micrograms per milliliter of 1F5-T15 was used to coat Costar vinylassay plates (Costar, Cambridge, Mass., U.S.A.). After blocking with 1%(BSA) solution, 8 ug/mL photobiotinylated (see U.S. Pat. No. 5,800,991discussed above) 1F5-T15 naked 1F5 and control antibody (5D10) wereadded, diluted to 1:1, and incubated for 2 hours at room temperature.After washing with PBS buffer, avidin-HRP (Sigma, St. Louis, Mo.,U.S.A.) was added, and enzyme-linked immunosorbent assay color wasdeveloped with o-phenylenediamine.

FACS Assay of the B-Cell Lymphoma

SU-DHL-4 cells were fixed using 1% paraformaldehyde, and 1×10⁶ cellswere suspended in 50 μL staining buffer (Hanks, containing 0.1% NaN3 and1.0% BSA); 1.5 μg photobiotinylated 1F5-T15 conjugates (see U.S. Pat.No. 5,800,991 discussed above), naked 1F5, and control antibodies wereadded and incubated for 30 minutes on ice. The cells were washed twicewith staining buffer, and then avidin-FITC was added for 30 minutes onice. After washing twice with staining buffer, the cells wereresuspended in 200 .mu.L PBS for FACS analysis.

Hoechst-Merocyanin 540 Staining to Detect Apoptosis

After 1×10⁶ lymphoma cells were placed into 24-well tissue culturewells, 4 μg antibodies and antibody-peptide conjugates were added. After24 hours of incubation, 1×10⁶ cells were removed from the culture pelletand resuspended in 900 μL cold PBS (pH 7.2), and 100 μL Hoechst (Pierce,Rockford, Ill., U.S.A.) 33342 (50 μg/mL) was added and incubated at 37°C. for 30 minutes in the dark. The cells were centrifuged andresuspended in 100 μL PBS; 4 μL MC540 dilution solution was added andthe cells were incubated for 20 minutes at room temperature in the dark.The cells were pelleted, resuspended in 1 mL PBS, and analyzed by flowcytometry.

Inhibition of Cell Growth in Culture

One×10⁵ tumor cells were seeded in complete culture medium. At days 1,2, and 3 of culture, aliquots were removed and viable cells were countedusing dye exclusion (trypan blue).

Results

Mouse monoclonal antibodies 1F5 IgG2a were conjugated with self-bindingpeptide as in Example 1. An average of 1.8 peptides were found bycompetitive analysis. The parental antibody was compared to theconjugated form for binding by flow cytometry. As shown in FIG. 3 thebinding was increased for the conjugated antibody (Mab-ap) when assessedwith a limiting dilution of antibody. This was characterized by a shiftin the binding fluorescence to a higher intensity. When compared over aseries of dilutions, conjugated antibody required almost one-tenth theconcentration of antibody to achieve the same level of intensity asparental antibody (FIG. 2). As shown in FIG. 1, increasing the amount ofconjugated antibody caused a reduction in fluorescence intensity,presumably due to internalization, a property of SAT technology that canbe used to enhance potency of immunoconjugates of drugs, toxins andshort path length radiotherapeutic isotopes. Furthermore, when testedfor the ability to trigger apoptosis, the conjugated form (Sab) was muchmore active than native antibody, with most cells dead by 3 days,compared to only a small fraction with the native antibody (see FIG. 4).

Example 4

Enhanced Binding and Apoptosis with Anti-GM2 Antibodies

Materials and Methods

Cell Lines and Antibody

Human T-cell leukemia Jurkat cells were grown in RPMI 1640 supplementedwith 10% fetal bovine serum and antibiotic (penicillin, streptomycin andamphotericin). Chimeric hamster anti-GM2 antibody (ch-α-GM2) wasobtained from Corixa Corporation (Seattle, Wash.). After chimerization,the resulting antibody lost its ability to induce apoptosis inganglioside GM2 expressing target cells.

Synthesis of Antibody-Peptide Conjugate

Both the T15 peptide (GAAASRNKANDYTTEYSASVKGRFIVSR) (SEQ ID NO. 8), aVH-derived peptide from a self-binding antibody-T15 (Kaveri et al,1991), and a scrambled peptide (T15-scr) (SEQ. ID. NO. 3), which wasrandomly generated using the T15 amino acid sequence, were synthesizedby Genemed Synthesis (South San Francisco, Calif.). The scrambledpeptide was used as a control. Antibodies were dialyzed against PBS (pH6.0), then 1/10 volume of 200 μM NaIO4 was added and incubated at 4° C.for 30 min in the dark. The reaction was stopped by adding glycerol to afinal concentration of 30 μM, and the samples were dialyzed at 4° C. for30 min against PBS (pH 6.0). Fifty (50) times molecular excess of T15 orscrambled peptide was added to the antibodies and incubated at 37° C.for 1 h. L-Lysine was added and incubated at 37° C. for 30 min to blockthe remaining reactive aldehyde group. After the blocking step, theantibody-conjugates were dialyzed against PBS (pH 7.2) at 4° C.overnight, then stored at 4° C. until used.

Direct Binding ELISA

GM2 ganglioside was dissolved in methanol and 0.5 μg was coated per wellin 96 well polystyrene plates (Costar, Cambridge, Mass.) and allowed todry overnight. The wells were blocked with 1% BSA for 2 h at roomtemperature and 400 μg of anti-GM2 antibodies, diluted in 1% BSA, wereadded in the first well and then serially diluted 1:1. After incubationfor 1 h, the wells were washed 5×. and HRP-conjugated anti-human IgG(Sigma, St. Louis, Mo.) was added at a 1:1000 dilution and incubated for1.5 h. After washing three times, the bound antibodies were visualizedusing substrate o-phenylenediamine and read at OD 492 using aspectrophotometer.

Specific Binding ELISA

Gangliosides GM2, GM1, GM3 were dissolved in DMSO in 0.5 μg and coatedin 96 well polystyrene plate (Costar, Cambridge, Mass.) dried overnight. The wells were blocked with 1% BSA for 2 h at room temperature,400 μg of ch-aα-GM2 antibodies (anti-GM2-T 15) were added in the firstwell and then serially diluted 1:1. After incubation for 1 h, the wellswere washed 5× and HRP-conjugated anti-human IgG (Sigma, St. Louis, Mo.)was added and incubated for 1.5 h. After washing three times, the boundantibodies were visualized using substrate o-phenylenediamine andassayed as described previously.

Antibody Self-Binding ELISA

2 μg/ml of naked ch-α-GM2 (anti-GM2) or ch-α-GM2-T15 (anti-GM2-T15) werecoated onto Costar vinyl assay plates. After blocking with 3% BSAsolution, 0.5 μg/well of photobiotinylated anti-GM2-T15 was added. Theantibodies were then incubated for 2 h at room temperature. Afterwashing three times, avidin-HRP (Sigma) was added at a 1:1000 dilutionand incubated for 1 hour. The bound antibodies were visualized by addingsubstrate o-phenylenediamine and assayed as described previously.

Cell Surface Binding Detected by FACS

Two×10⁵ Jurkat cells per well were seeded in a 6-well plate andincubated overnight, then cells were collected and washed twice withP/B/G/A buffer (0.5% BSA, 5% Goat Serum in PBS). Cells were thenresuspended in 100 μl P/BIG/A buffer containing 5 μg/ml anti-GM2antibodies for 30 min. After washing with P/BIG/A buffer,FITC-conjugated anti-Human IgG (Sigma, 1:1000 dilution in 100 μlP/BIG/A) was added and incubated on ice for 30 min. After washing with.P/BIG/A buffer, cells were resuspended in 400 .mu.l P/B/G/A containing10 μg/ml propidium iodide (as viability probe) and analyzed by flowcytometry.

Apoptosis Detected by Annexin V Staining

2×10⁵ Jurkat cells were seeded per well in a 6-well plate. After 6 h,cells were incubated with 20 μg/ml of the anti-GM2 or anti-GM2-T15antibodies for 12 hr. Following the incubation, a small portion of cells(50 μl) was saved and assayed for viability, while the remainder of thecells were harvested and washed with cold PBS. Cells were thenresuspended in 100 μl annexin staining buffer (5 .mu.l Alex fluor 488was added into 95 μl l.times.annexin binding buffer, and Sytox was addedat a dilution of 1:1000. After incubation at room temp for 15 min, 400μl of 1xannexin binding buffer was then added, and samples were analyzedby FACS.

Viability Assay for Antibody-Treated Cells

A small portion of the cell samples saved from the annexin experimentwas used for viability assay. 10-μl aliquots from the cell suspensionwere taken to determine viability using trypan blue exclusion assay.

Statistical Analysis.

Statistical analysis was performed using one-way ANOVA followed byNewman-Keuls post test. Data are reported as means±SD.

Results

Self-Binding Peptide Enhanced Antibody Binding to its SpecificGanglioside.

Following antibody-peptide conjugation, the binding capacity of theT15-conjugated ch-α-GM2 antibody (anti-GM2-T15) was determined using adirect binding ELISA. As seen in FIG. 9, both ch-α-GM2 antibody(anti-GM2) and anti-GM2-T15 antibody showed a dose-dependent increase inbinding to ganglioside GM2. The anti-GM2-T15 antibody demonstrated ahigher binding capacity compared with the naked anti-GM2 at all thedoses tested, confirming that the self-binding T15 peptide had increasedthe antigen binding capacity of the ch-α-GM2 antibody at a givenantibody concentration.

Antibody Self-Binding Behavior Demonstrated by ELISA

Next, it was investigated by ELISA whether the increase in binding toganglioside GM2 by the T15 peptide-linked antibody was due to itsself-binding feature. As seen in FIG. 10, the anti-GM2-T 15 antibodydemonstrated a greater dose-dependent increase in binding to thepeptide-conjugated anti-GM2-T15 antibody coated on the wells, whereas itdid not show significant binding to the non-peptide conjugated anti-GM2antibody. These data demonstrate that the anti-GM2-T15 antibody can bindto itself or homodimerize through the Fc-conjugated, autophilic peptidemoiety.

15 Conjugation does not Change the Specificity of the ch-α-GM2 antibody.

To assess whether conjugation of the T15 peptide might alter the cognatebinding specificity of the antibody, a direct antigen-binding ELISA wasused to determine the binding specificity of the anti-GM2-T15 conjugatedantibody. As shown in FIG. 11, the anti-GM2-T15 antibody demonstrated aspecific, dose-dependent increase in binding to ganglioside GM2, whereasno binding above background levels to gangliosides GM1 or GM3 wasdetected. This result confirms that addition of the self-binding T15peptide did not alter nor reduce the specificity of the ch-.alpha.-GM2antibody.

Enhanced Surface Binding of Anti-GM2 Antibody to Target Tumor Cells

The human T-cell leukemic cell line Jurkat is known to expressganglioside GM2 (Suzuki et al, 1987). The ability of thepeptide-conjugated anti-GM2-T15 antibody to bind to native gangliosideGM2 expressed on the surface of Jurkat cells was compared to that of thenon-conjugated anti-GM2 antibody by flow cytometry. As shown in FIG. 12,the ch-.alpha.-GM2 antibody (anti-GM2) demonstrated a GM2 specificbinding signal 3 times greater than background levels, whereas thebinding demonstrated by the T15-conjugated anti-GM2 antibody was 2-foldhigher than that of the non-peptide conjugated antibody. This resultsuggests that the enhanced binding demonstrated by thepeptide-conjugated Ab is due to self-aggregation of this antibody.

Inhibition of Tumor Growth

Antibodies binding to the B cell receptor have been shown to inducecrosslinking of the BCR, which, in turn, inhibits cell proliferation(Ward et al, 1988) and produces a death signal (Hasbold et al, 1990;Wallen-Ohman et al, 1993). Furthermore, chemically dimerized antibodiesdirected against a B-cell tumor induce hyper-crosslinking of the BCRfollowed by inhibition of cell division and induction of apoptosis ofthe tumor cells (Ghetie et al, 1994; Ghetie et al, 1997). To determinewhether the T15-conjugated anti-GM2 antibody induced a similaranti-proliferative effect, 2×10⁵ Jurkat cells were cultured in thepresence or absence of anti-GM2 or control antibodies for 12 h, and thenthe number of viable cells remaining were counted. As summarized in FIG.13, no antibody or control human IgG antibody (HuIgG) treatment had noeffect on cell growth or viability, whereas there was some effect withthe anti-GM2 antibody. However, the T15-linked antibody demonstrated amarked inhibition of Jurkat cell growth, as cell numbers werereduced >2-fold compared to naked anti-GM2 antibody treated cells, andmore than 4 fold versus the control IgG treatment. As a comparison andpositive control, Actinomycin D demonstrated the ability to induceapoptosis, at levels slightly higher than the SuperAntibody.

Induction of Apoptosis

In order to determine whether the anti-tumor effect of antibodiesdirected against cell surface expressed gangliosides might be due to theinduction of apoptosis, we took the cell samples used in the cell growthstudy and analyzed them for apoptosis induction by measuring annexin Vstaining. The results are summarized in Table I.

TABLE I Apoptosis Analysis Using Annexin V Staining Antibody Jurkat* Notreatment  7.7 ± 1.55 HuIgG  7.2 ± 1.94 Anti-GM2 14.8 ± 7.55Anti-GM2-T15scr 13.0 ± 4.60 Anti-GM2-T15 54.2 ± 23.4 Actinomycin D 81.9± 10.2 *Data were summarized from four sets of experiments.

Treatment of Jurkat cells with the ch-α-GM2 antibody (anti-GM2) or thech-α-GM2 antibody conjugated with a scrambled, control peptide(anti-GM2-T15scr) did not induce apoptosis significantly over levelsinduced by treatment with control human IgG, as a modest 2-fold increasewas observed. However, Jurkat cells treated with the anti-GM2-T15conjugated underwent a significant amount of apoptosis, nearly 8-foldover background and more than 4-fold higher than that induced by thenon-conjugated antibody or the control-conjugated antibody. Theseresults confirmed the activity and specificity of T15-conjugatedantibody.

Example 5

Generation of Autophilic Peptide Sequences T15-scr, T15-scr2, R24 andR24-Charged

Peptides were synthesized as in Example 5. The sequences are given inTable II.

TABLE II Sequences for Autophillic Binding Peptides NameSequence (NH2 to COOH) T15 ASRNKANDYTTDYSASVKGRFIVSR  (SEQ ID NO. 1)T15scr  SYSASRFRKNGSIRAVEATTDVNSAYAK  or T15s (SEQ ID NO. 3) T15scr2SKAVSRFNAKGIRYSETNVDTYAS  (SEQ ID NO. 4) R24 GAAVAYISSGGSSINYA (SEQ ID NO. 5) R24- GKAVAYISSGGSSINYAE  Charged (SEQ ID NO. 6)

The peptide derived from R24 is difficult to solubilize except in DMSOor alcohol. Using such solubilizers can not only denature the antibodybut also makes it difficult to conjugate to hydrophilic regions of theantibody. To overcome this solubility problem the addition and changesof sequence to charged amino acids, as shown in Table II wereundertaken. The resultant modified peptide (R24-Charged) was soluble inaqueous buffer, was able to be conjugated to the tryptophane ornucleotide binding sites and preserved self-binding as well as inducedapoptosis when conjugated to anti-GM2 antibody. The same amino acidspresent in the T15 sequence were randomly re-arranged and used toconstruct a further synthetic peptide; this scrambled sequence (T15scror T15s), had no self-binding and when conjugated to anti-GM2 antibodydid not induce apoptosis (see Example 5, Table I). In like manner, asecond, randomly selected sequence, derived from the amino acids of theT15 sequence, was used to generate a synthetic peptide (T15scr2). Unlikethe first scrambled sequence, this peptide demonstrated self-binding andwhen conjugated to anti-GM2 antibody, induced apoptosis in levels higherthan the original T15 sequence. Thus, self-binding behavior can begenerated, using the same amino acids from the original T15 sequence butarranged in a different order from the original T15. A peptide librarygenerated using these same amino acids, combined with a screen forself-binding could be used to identify other self-binding sequences.

Example 6

Method of Conjugating Autophilic Peptides to Antibodies (Comparison ofVarious Immunoglobulin Conjugation Sites)

The T15 peptide sequence was conjugated to anti-GM2 antibody via thenucleotide binding site, tryptophane affinity sites, and throughperiodate oxidation, the carbohydrate on the Fc region. As shown in FIG.6, when tested for the ability to trigger apoptosis, the nucleotide siteconjugation (GM2-N-3-ATP-T15/biotin) generated a higher level ofapoptosis, than the carbohydrate linkage (Anti-GM2-T15). This was inspite of the fact that carbohydrate linkage installed 8-10 peptides perantibody and nucleotide linkage only 2 peptides per antibody. Affinitysite conjugation was the best method of conjugation of peptides.Conjugation to epsilon-amino acids of antibody, via hetero-bifunctionalcross-linking agents, gave an inactive conjugate (not shown).

Example 7 Restoration of Apototic Activity

A parental antibody to GM2 glycolipid, derived from a non-humanhybridoma, was tested for the ability to trigger apoptosis against humancancers including non-small cell lung cancer (FIG. 5). The parentalantibody demonstrated a high level of apoptosis and killing of cancercells. The antibody was also effective in inhibiting growth of cancersin nude mouse models (not shown). To remove the potential forimmunogenicity in humans, the antibody was “humanized” via cloning theheavy and light chain CDR's into the context of a human IgG1. Despiteretention of affinity and specificity (not shown), the humanizedantibody demonstrated much reduced ability to trigger apoptosis. Incontrast, the humanized antibody, conjugated to a self-binding peptide(Sab), demonstrated high levels of apoptosis, similar to that of theparental antibody.

A further example is of a murine antibody, R24 which targets the GD3ganglioside on human melanoma cells. When naturally expressed, thisantibody has self-binding and therapeutic activity in patients, but as ahumanized antibody it loses avidity, self-binding and therapeuticactivity (Chapman et al., 1994). Restoration of therapeutic activity ofthe humanized R24 antibody can also be achieved by conjugation of aself-binding peptide to the antibody.

The humanized versions of antibody TEPC-15 and T15/S107 will alsobenefit from conjugation with a self-binding peptide to restore orenhance self-binding and therapeutic activity.

Example 8 Enhanced Binding and Tumor Recognition by HerceptinSuperAntibody

Herceptin (monoclonal antibody to HER2/neu), has been approved by theFDA for treatment of breast cancer. The antigen is expressed inapproximately 30% of breast cancers but in only about half of thosepatients is the level of expression sufficient to trigger therapeuticeffects. In fact, patients are normally pre-screened in a diagnostictest to determine their suitability for treatment. HER2/neu is alsoexpressed on other cancers, such as non-small cell lung cancer buttypically in only low levels, making this type of cancer unsuitable fortreatment. We conjugated an autophillic peptide to Herceptin and testedfor ability to bind non-small cell lung cancer. As shown in FIG. 8 (toppanel), Herceptin reacts very weekly to this cancer; only 0.5% of cellsare positive compared to an irrelevant antibody. In contrast, the samecancer can be better detected with the autophilic peptide conjugatedform (i.e. SuperAntibody form) of Herceptin; over 57% are positivecompared to irrelevant antibody (bottom panel). In separate tests, aSuperAntibody form of Herceptin also inhibited growth better than theparent antibody and could trigger apoptosis unlike the parent.

As will be apparent to those skilled in the art in the light of theforegoing disclosure, many alterations and modifications are possible inthe practice of this invention without departing from the spirit orscope thereof. Accordingly, the scope of the invention is to beconstrued in accordance with the substance defined by the followingclaims.

REFERENCES

The pertinent disclosures of the following references are incorporatedherein by reference:

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1. An autophilic antibody comprising an autophilic peptide conjugated toan immunoglobulin component of a non-autophilic antibody.
 2. Anautophilic antibody according to claim 1, wherein the autophilic peptidecomprises a conserved self-binding sequence.
 3. An autophilic antibodyaccording to claim 1, wherein the autophilic peptide is a humanizedpeptide.
 4. An autophilic antibody according to claim 1, wherein theautophilic peptide is selected from the group consisting of T15 peptide,T15-scr2 peptide, R24 peptide, and R24-Charged peptide.
 5. An autophilicantibody according to claim 1, wherein the autophilic peptide comprisesa peptide selected from the group consisting of: SEQ ID NO.1; SEQ IDNO.4; SEQ ID NO.5; and SEQ ID NO.6.
 6. An autophilic antibody accordingto claim 1 comprising one or more functional peptides conjugated to thenon-autophilic antibody.
 7. An autophilic antibody according to claim 6,wherein the one or more functional peptides are conjugated to thenon-autophilic antibody at a site that is different from a site that theautophilic peptide is conjugated to the non-autophilic antibody.
 8. Anautophilic antibody according to claim 6, wherein the one or morefunctional peptides comprises an MTS peptide.
 9. An autophilic antibodyaccording to claim 6, wherein the one or more functional peptidescomprises a peptide selected from the group consisting of: SEQ ID NO.2;and SEQ ID NO:
 7. 10. An autophilic antibody according to claim 1,wherein the non-autophilic antibody is selected from the groupconsisting of 5D10, S1C5, rituximab, anti-GM2, trastuzumab, anti-caspaseantibodies, humanized TEPC-15, humanized R24, and humanized S107 orwherein the autophilic antibody is selected from the group consistingof: S1C5 antibody conjugated with T15 peptide; 5D10 antibody conjugatedwith T15 peptide; anti-caspase 3 antibody conjugated with MTS peptide;anti-CD20 antibody conjugated with T15 peptide; rituximab conjugatedwith T15 peptide; IF5 conjugated with T15 peptide; tositumomabconjugated with T15 peptide; anti-GM2 conjugated with T15 peptide; anon-human antibody to GM2 glycolipid conjugated with an autophilicpeptide; and Herceptin conjugated with an autophilic peptide.
 11. Amethod of producing an autophilic antibody according to claim 6comprising expressing the autophilic antibody as a fusion proteincontaining the autophilic peptide sequence, the one or more functionalpeptides, and the non-autophilic antibody.
 12. A composition comprisingone or more autophilic antibodies according to claim 1 and apharmaceutically acceptable carrier.
 13. A composition comprising one ormore autophilic antibodies according to claim 6 and a pharmaceuticallyacceptable carrier.
 14. A method of treating a patient suffering from adisease or disorder responsive to antibody therapy comprisingadministering to the patient one or more autophilic antibodies accordingto claim 1, or a composition comprising one or more autophilicantibodies according to claim 1 and a pharmaceutically acceptablecarrier, in a therapeutically effective amount to alleviate symptoms ofthe disease or disorder.
 15. The method of claim 14, wherein the diseaseor disorder is a malignancy, an auto-immune disorder, transplantationrejection, Alzheimer's disease, a neuro-degenerative condition,atheroschlerosis, or any other condition responsive to antibody therapy.16. The method of claim 14, wherein the autophilic antibody isadministered in one or more dose amounts substantially identical toamounts used to administer non-autophilic antibodies.
 17. The method ofclaim 14, wherein an initial dose is about 250 mg per day and a laterdose is about 100 mg per week.
 18. The method of claim 14, wherein amaintenance dose is about 50 mg per week.
 19. A method of claim 14,wherein the treating comprises potentiating apoptosis, complementfixation, or cell-mediated killing of selected cells in the patient. 20.A method according to claim 19, further comprising administering to thepatient a second antibody directed to the autophilic peptide of theautophilic antibody.